Comparative analysis of POD genes and their expression under multiple hormones in Pyrus bretschenedri

Background Class III peroxidase (POD) enzymes play vital roles in plant development, hormone signaling, and stress responses. Despite extensive research on POD families in various plant species, the knowledge regarding the POD family in Chinese pear (Pyrus bretschenedri) is notably limited. Results We systematically characterized 113 POD family genes, designated as PbPOD1 to PbPOD113 based on their chromosomal locations. Phylogenetic analysis categorized these genes into seven distinct subfamilies (I to VII). The segmental duplication events were identified as a prevalent mechanism driving the expansion of the POD gene family. Microsynteny analysis, involving comparisons with Pyrus bretschenedri, Fragaria vesca, Prunus avium, Prunus mume and Prunus persica, highlighted the conservation of duplicated POD regions and their persistence through purifying selection during the evolutionary process. The expression patterns of PbPOD genes were performed across various plant organs and diverse fruit development stages using transcriptomic data. Furthermore, we identified stress-related cis-acting elements within the promoters of PbPOD genes, underscoring their involvement in hormonal and environmental stress responses. Notably, qRT-PCR analyses revealed distinctive expression patterns of PbPOD genes in response to melatonin (MEL), salicylic acid (SA), abscisic acid (ABA), and methyl jasmonate (MeJA), reflecting their responsiveness to abiotic stress and their role in fruit growth and development. Conclusions In this study, we investigated the potential functions and evolutionary dynamics of PbPOD genes in Pyrus bretschenedri, positioning them as promising candidates for further research and valuable indicators for enhancing fruit quality through molecular breeding strategies. Supplementary Information The online version contains supplementary material available at 10.1186/s12863-024-01229-7.


Background
Peroxidases (PODs) occur in a wide range of living organisms and are considered a diverse multigene family [1,2].With the use of hydrogen peroxides as an electron acceptor in their active center with a metal, peroxidases are known to catalyze oxidative reactions [3].Heme PODs, along with nonheme PODs are two major groups of peroxidases based on variations in their structure.Two subfamilies (e.g., animal PODs, alongside nonanimal PODs) comprise the heme PODs whereas three major classes viz., class I, class II, and class III, comprise the nonanimal superfamily [1,4].Class III peroxidases act as plant-specific oxidoreductases and various studies have abbreviated the class III peroxidases in various ways, such as PER, Px, POX, Prx, and POD [5].POD is the abbreviation that is used in this study, and it is basically a plantspecific oxidoreductase.PODs are widely distributed in microorganisms, plants, and animals [2].In terms of plant growth, PODs are known for their dual function, as they can both harden and soften plant cell walls, and have been reported to play roles various processes, such as germination, lignification, development as well and plant defense via various action mechanisms, for example the formation of radicals, the regulation of ROS, and substrate oxidation [3].
With the advent of transcriptomic analysis, large numbers of PODs, which are known to perform different functions, have been identified.The role of PODs is still elusive, with only a few studies present in the literature highlight the functional role of PODs [7].Moreover, cold stress resistance improved with the POD (AtPrx69, AtPrx22, and AtPrx39) gene overexpression in Arabidopsis thaliana [7].However, the POD gene in cotton, namely GhPOX1 is known for its ability to increase ROS production [8].The regulation of the POD genes in Zea mays (roots) are regulated by salicylic acid, methyl jasmonate, and pathogen elicitors [9].According to various studies, POD genes are primarily involved in resisting or responding to stress stimuli in addition to playing some physiological and biological roles [10].
Bioinformatics analysis has been majorly used to study and characterize the number of POD in various, plants including 73 PODs in Arabidopsis thaliana; 93 in Populus trichocarpa, 138 in one of the important serial crop species, e.g., Oryza sativa; 119 in Zea mays; and 102 in Medicago sativa [3,11].The demand for pear (Pyrus species) fruit demand has increased around the world due to its low price and health benefits [12].A wide range of bioinformatics analysis were performed in this study for the POD gene family; moreover, these genes play pivotal roles in helping plants respond to or resist various stress stimuli.In total, 113 genes were identified for the first time in the pear genome, and this analysis was performed with the aid of genome-wide approaches.
In this study, we have considered chromosomal mapping, physicochemical properties, gene duplication events, phylogenetic relationships, collinear correlation, rate of substitution, GO and KEGG enrichment analysis, promoter sequence analysis, and expression profiling in response to various conditions under melatonin (MEL), salicylic acid (SA), abscisic acid (ABA), and methyl jasmonate (MeJA) stress.The present study contributes to the future enhancement of crop and fruit quality, providing a deeper understanding of the various POD genes.Research on pears is of utmost importance because it lays the foundation for improving the cultivation of this fruit.Therefore, the results obtained in this study may lead to advancements in the characterization of this species/ genus, ultimately benefiting fruit quality.Our research is designed to offer a comprehensive global classification and analysis of plant gene families.

Results
POD gene family identification and characterization in P. bretschneideri.In this study, we identified a total of 113 POD genes within the P. bretschneideri genome, for simplicity, we designated them as PbPOD1 through PbPOD113 based on their corresponding chromosomal position.Additionally, we delved into valuable details about these PODs, including their protein identifiers, where they are located on the chromosomes, the length of their coding sequences (CDS) in base pairs, and various physical attributes, such as isoelectric points (pIs), molecular weight in kilodaltons (kDa), as well as protein length in amino acids (aa), and the grand average of hydropathicity (GRAVY).
While protein length varied between 83 amino acids (PbPOD36) and 1314 amino acids (PbPOD66) with an average of 335.22 amino acids.Similarly, Molecular weight ranged between 9017.36 kDa (PbPOD36) and 143415.68kDa (PbPOD66) with a mean value of 36.61 kDa.On the other side, isoelectric points ranged between 4.29 (PbPOD11) and 9.73 (PbPOD35).The GRAVY results displayed diversity, with values spanning from − 1.001 (PbPOD30) to 0.124 (PbPOD14).It is noteworthy that most of these genes exhibited hydrophilic properties, with 15 genes demonstrating hydrophobic characteristics by displaying positive GRAVY values (Table 1).

Phylogenetic relationship of PbPOD gene family
To explore the evolutionary relationships among the POD family genes, we generated a comprehensive phylogenetic tree of the 113 PbPODs and 73 AtPODs of Arabidopsis thaliana using the maximum likelihood method in MEGA 7.0.The phylogenetic tree revealed that PODs can be additionally classified into seven distinct subgroups (Fig. 1).The findings demonstrated an Our analytical outcomes segmented the POD genes into five subfamilies, namely, I, II, III, IV, V, VI and VII.
For a comprehensive exploration of the phylogenetic affiliations and potential functional divergence inherent to POD genes, homologs from both the P. bretschneideri and A. thaliana genomes were selected, which enabled a multifaceted sequence alignment and a subsequent analysis of the phylogenetic architecture, as illustrated in Fig. 1.Considering the variances in protein structural configurations, in the present study, we divided the POD family into five highly conserved, distinct subfamilies.Each subfamily was confirmed to be robust through rigorous bootstrap validation.Based on phylogenetic assessment, the POD genes were taxonomically classified into  ).An ancillary aspect of our research centered on the phylogenetic ties of the POD genes of P. bretschneideri with those of in A. thaliana.Conclusive evidence suggested that the POD genes from both of these species demonstrated a closely intertwined evolutionary trajectorie, as shown in Fig. 1.

Chromosomal localization of PbPOD genes
Chromosomal mapping of PbPOD genes was conducted based on the available genome assembly of P. bretschenedri.In total, these genes are distributed across 17 chromosomes, displaying notable disparities in gene density across different chromosomal regions.Specifically, chromosomes 3, 7 and 8 harbor a relatively high density of PbPOD genes (Fig. 2).By identifying these uneven distribution patterns and gene clusters, this study sheds light on the underlying genomic architecture that may have implications for the functional specialization and evolutionary history of the PbPOD gene family in P. bretschenedri.The presence of clustered PbPOD genes possibly indicates regions of the genome that have undergone tandem duplication events, which may, in turn, play a role in the rapid diversification and functional expansion of this gene family (Fig. 2).
The findings pertaining to the chromosomal localization of PODs demonstrated heterogeneous distribution patterns, with protein quantities varying from one to seven per chromosome, excluding chromosome 1, which had the lowest number of genes in each species.These distribution patterns were observed across a total of 17 distinct chromosomes, specifically Chr1 to Chr17, within Fig. 2 Genomic localization and distribution patterns of PbPOD genes in the P. bretschenedri genome the genome of the pear.Furthermore, the chromosomes exhibited varying gene counts, with Chr7 displaying an impressive number of genes, such as P. bretschenedri vs. P. persica 13 gene pair clusters, P. bretschenedri vs. P. avium 9, P. bretschenedri vs. P. mume 12 pairs and P. bretschenedri vs. F. vesca has 16 pairs (Fig. 3 and Table S2).In addition, in P. bretschenedri and P. avium; P. bretschenedri and F. vesca, exhibited 73 and 86 pairs, respectively.These findings align with the established evolutionary relationships among these species.Hence, within the POD members, notable patterns of genetic variation were detected in the genome of the pear.To gain a deeper understanding of the evolutionary patterns of PbPOD genes during the evolutionary process, we investigate more extensive synteny blocks in P. bretschenedri.According to collinearity analysis of the PbPOD gene, a total of 54 gene pairs were identified to be involved in the replication event (Fig. 4).
Throughout the course of evolutionary events, the genetic elements experience a multitude of selection pressures, encompassing positive selection (with a Ka/ Ks ratio greater than 1), purifying selection (with a Ka/ Ks ratio less than 1), and neutral selection (with a Ka/Ks ratio equal to 1).The gene duplications of 113 PbPOD family members were analyzed.These pairs can be categorized into different types, including 15 pairs that were proximal, 4 pairs that were transposed, 32 pairs of segmental duplications, and 3 pairs that were tandem duplications (Table S3).The experimental findings indicated that the majority of the gene pairs exhibited a Ka/Ks ratio of less than 1.00 (Table S3), implying the presence of purifying selection.This observation further unveiled a restricted level of divergence after duplications of genes.
Fig. 3 Analysis of syntenic relationships in the POD gene family between P. bretschenedri and Selected Rosaceae Species: P. avium, P. mume, P. persica and F. vesca study explores the syntenic relationships of the POD gene family between P. bretschenedri (Pb) and four other Rosaceae species-P.avium (Pv), P. mume (Pm), P. persica (Pp) and F. vesca (Fv).The analysis employs Bezier lines as a graphical representation to identify and delineate the collinear blocks of genes shared between the two species being compared.These lines serve as the background framework upon which specific gene pairs are mapped However, it was observed that most of Ka/Ks with values less than 0.6 (Table S3), indicating PbPOD gene family may undergo strong negative selection during evolution.

GO and KEGG and cis-regulatory elements analysis in pear
The GO enrichment analysis was conducted to elucidate the functional regulatory mechanism of POD genes.The observation revealed the presence of three distinct subgroups, namely cellular components, molecular functions, and biological processes (Fig. 5).In the BP processes, the GO terms hydrogen peroxides catabolic process (GO:0042744), response to oxidative stress (GO:0006979); cellular oxidant detoxification (GO:0098869); cellular oxidant detoxification (GO:0098869); hydrogen peroxide catabolic process (GO:0042744); response to oxidative stress (GO:0006979); and cellular oxidant detoxification (GO:0098869) exhibit significant enrichment.In a similar manner, the GO terms associated with CC processes and MF primarily pertain to molecular components such as lactoperoxidase activity (GO:0140825); heme binding (GO:0020037); metal ion binding (GO:0046872); heme binding (GO:0140825); metal ion binding (GO:0046872).In the cellular component, extracellular region (GO:0005576); plant-type cell wall (GO:0009505); membrane (GO:0016020); extracellular region (GO:0005576); vacuole (GO:0005773).The GO terms for molecular function (MF), cellular component (CC), and biological process (BP) indicate the significant involvement of PODs in diverse grapevine activities.Furthermore, the KEGG enrichment analysis revealed the presence of three prominent pathways within the grapevine's PODs, namely "Biosynthesis of other secondary metabolites, " "Phenylpropanoid biosynthesis, " and "Metabolism" (Table S4).
Furthermore, the cis-acting elements located in the promoter region of POD members were analyzed utilizing the PlantCARE database.In a concise manner, the  indirect participation across multiple biotic and abiotic hormone signaling pathways (Fig. 6 and Table S5).

Analysis of POD gene expression in different organs of pear
In the current study, the investigation focused on the expression profiling of all 113 PbPOD genes in pear.These PODs were derived from 6 different organs and tissues (stem, leaf, bud, ovary, petal, and sepal), and their expression patterns were analyzed.The RNA-seq data were obtained from the NCBI database.To depict the spatiotemporal expression pattern, a graphical representation in the form of a heatmap was constructed (Fig. 7).This heatmap was based on the FPKM values, which were logarithmically transformed of the 113 PbPOD (P.bretschenedri Peroxidase) genes.The specific details of these genes can be found in Table S6.The expression levels of 16 PbPOD genes (PbPOD4, PbPOD5, PbPOD11, PbPOD17, PbPOD26, PbPOD28, PbPOD34, PbPOD38, PbPOD53, PbPOD75, PbPOD84, PbPOD89, PbPOD93, PbPOD97, PbPOD108, and PbPOD112) exhibited significant and highest expressions in stem, and 10 PbPOD (PbPOD1, PbPOD2, PbPOD7, PbPOD10, PbPOD24, PbPOD43, PbPOD66, PbPOD71, PbPOD90, and PbPOD96) genes were highly expressed in leaves, while six genes, PbPOD3, PbPOD9, PbPOD12, PbPOD13, PbPOD46, and PbPOD72 were highly expressed in buds.In addition, some PbPOD genes were significantly expressed in ovary, petal, and sepal, such as PbPOD10, PbPOD55, and PbPOD80.These results indicated that their crucial functions in pear.As well as 27 genes out of 113 no significant expression in any stage.On the other hand, some genes showed lower and no significant expression (PbPOD41, PbPOD52, PbPOD64, PbPOD66, PbPOD78, and PbPOD106) but after hormonal treatment these showed higher expression of different time interval.These results imply their genes have potential involvement in stem, leaf, bud, ovary, petal, and sepal and fruit development stages.Furthermore, the remaining genes exhibited either moderate or weak levels of expression abundance in all the chosen tissues and organs, suggesting their restricted responsiveness in pear plants.

Expression of PbPOD genes during fruit development and under abiotic stress
To explore the involvement of PbPOD genes in different developmental stages involving 15 DAF, 39 DAF, 47 DAF, 55 DAF, 63 DAF, 79 DAF, 102 DAF, and 145 DAF qRT-PCR expression analysis was performed.In the ontogenetic stages of Pyrus bretschneideri fruit, the expression patterns of the PbPOD genes exhibited heterogeneity (Fig. 8).Specifically, PbPOD10, PbPOD52, and PbPOD64 demonstrated an upward transcriptional trajectory, peaking at 55 DAF, followed by a subsequent decrease.Conversely, PbPOD50 and PbPOD78 exhibited peaks at 47 DAF, and PbPOD41 exhibited a peak at 79 DAF, respectively, while PbPOD51, PbPOD80, PbPOD85, and PbPOD106 exhibited a peak at 145 DAF.Taken together, these results collectively suggest that POD gene Fig. 6 Cis-acting element analysis of PbPOD dene promoters: characterization and representation of diverse regulatory elements.Visualization of cis-acting elements in PbPOD promoters.The study conducted an extensive analysis to characterize cis-acting elements within the promoters of PbPOD genes.These elements are crucial for understanding the regulation of gene expression.In the graphical representation, different types of cis-acting elements are denoted by varying colors, as outlined in the color key provided on the left-hand side of the figure family members may play a putative role in modulating the development and growth processes of pear fruit.
We also explored the involvement of PbPOD genes in various abiotic stresses, including exposure to melatonin (MEL), salicylic acid (SA), abscisic acid (ABA), and methyl jasmonate (MeJA) via qRT-PCR analysis and selected 14 genes based on phylogenetic analysis.These genes were exposed to MEL, SA, MeJA and ABA stress treatments.The findings revealed that all these genes exhibited diverse responses, manifesting either low, high, Fig. 7 Heatmap clustering analysis in PbPOD genes expression in stem, leaf, bud, ovary, petal, and sepal).The scale bar serves as a visual guide for interpreting the degree and direction of gene expression changes.Moreover, the FPKM normalization method ensures that the expression levels are comparable across different genes and treatment durations or moderate expression levels in comparison to the control conditions.Under conditions of hormonal stress, it was observed that all genes exhibited increased expression levels.Subsequent to the exogenous hormonal treatments of SA, MeJA, and ABA at intervals of 1 h, 2 h and 3 h, and of MEL at 1 h, 4 h, and 16 h a marked variability in the transcriptional profiles of POD genes was observed, as shown in Figs. 9, 10, 11 and 12.
In Pyrus bretschneideri fruit subjected to ABA treatment, a substantial upregulation in the expression of PbPOD10 and PbPOD86 were evident just one-hour post-application, exhibiting fold-increases of 36.78 and 18.62, respectively, in comparison to the control at 0 h.Additional results indicated that the expression of PbPOD41, PbPOD66, PbPOD84, and PbPOD106 reached their maximum at 2 h, PbPOD17, PbPOD50, and PbPOD64 reached their highest at 3 h, with foldincreases of 14.01, 56.38, and 52.01, respectively (Fig. 9).
In the cohort treated with MeJA, a pattern analogous to that observed with ABA was discerned.Specifically, PbPOD55 was notably upregulated within an hour of treatment, displaying fold-changes of 3.71.Moreover, the expression levels of PbPOD52 and PbPOD66 peaked at the 2 h interval, with fold-changes of 18.7 and 26.13, respectively, in contrast to control.Finally, PbPOD10, PbPOD17, PbPOD41, PbPOD50, PbPOD51, PbPOD64, and PbPOD84 demonstrated a peak in expression at 3 h, In MEL treatment group, the expression level of PbPOD50, PbPOD64, PbPOD66, PbPOD78, and PbPOD106 reacheed their maximum at 1 h, while the expression levels of PbPOD41 reached its highest at 2 h.Simultaneously, PbPOD10, PbPOD17, PbPOD52, PbPOD80, and PbPOD84 manifested peak expression at the 3 h mark, with fold-changes of 43.12, 31.02,49.28, 79.31, and 41.59, respectively, relative to the control levels.Conversely, the expression of PbPOD86 was conspicuously inhibited among 1, 2, and 3 h post-treatment as compared to ABA and MeJA as shown in Fig. 11.
In the SA treatment group, expression profiles revealed that PbPOD51 experienced significant upregulation at 1 h post-application, and reached the highest level with fold-increases of 26.38 higher than the control.At the 2 h mark, the transcriptional activity of PbPOD64 reached its apex, with fold-changes of 56.58.Furthermore, at 3 h post-treatment, PbPOD10, PbPOD17, PbPOD52, PbPOD55, and PbPOD84 displayed peak expression levels, with respective fold-increases of 26.23, 10.82, 4.59, 14.01 and 75.85, as demonstrated in Fig. 12. Besides, compared with the control group, there was no significant change in the expression levels of PbPOD78 and PbPOD86 during the entire treatment process.Furthermore, an analysis of the correlation based on relative expression revealed a predominantly strong positive correlation, although some genes exhibited an inverse correlation.In summary, these results highlight the differential expression patterns of POD genes in response to multiple stressors, underscoring their potential significance in promoting plant growth and resilience.

Discussion
Due to the substantial role of Class III PODs in various physiological processes, including their involvement in responding to both biotic and abiotic stresses [1,13].Therefore, this is very necessary to systematic exploration of the potential functions of POD genes in pear, a crucial crop.In this research study, we successfully identified 113 PbPOD genes within the pear genome.This observation indicated that pear possesses more POD members compared to some reported species, such as Arabidopsis (73) [14] and Daucus carota (102) [1].However, a smaller Fig. 12 The relative expression of the PbPOD gene in response to salicylic acid (SA) hormonal stress was measured using quantitative PCR (qPCR).The results were normalized using an internal control, specifically the tubulin gene.The standard error (SE) represented by the error bars is based on three biological replicates.**Signifcant diference (P < 0.01), *signifcant diference at P < 0.05 Fig. 11 The relative expression of the PbPOD gene in response to melatonin (MEL) hormonal stress was measured using quantitative PCR (qPCR).The results were normalized using an internal control, specifically the tubulin gene.The standard error (SE) represented by the error bars is based on three biological replicates.**Signifcant diference (P < 0.01), *signifcant diference at P < 0.05 count compared to other species, such as rice (138) [13], tobacco (310) [15], and wheat (374) [16].Thus, this indicates a significant expansion of the POD gene family in pear, tobacco, and wheat compared to other plant species.
During plant evolution, gene duplications have signifcant impact on the expansion of gene families [17].In this study, we found that PbPOD genes are usually distributed in clusters on chromosomes, forming multiple gene clusters (Fig. 2), which is similar to the distribution of PpPOD genes on peach chromosomes [18].For example, chromosomes 3 and 7 contained 13 and 11 PbPOD genes, respectively (Fig. 2), which are adjacent and closely arranged, and have a very close evolutionary relationship with each other.In addition, we identified 54 duplication gene pairs from a total of 113 PbPODs (Fig. 3, Table S3).We believe that segmental duplication was considered the main driving force for the expansion and evolution of the PbPOD gene family.For example, 63% (34) of duplicated gene pairs were observed to be caused by segmental duplication, and 3 pairs (6%) of duplication pairs evolved through tandem duplication events.Our research findings are similar to the previous reports that segmental duplication is the main driving force for the evolution and expansion of the POD gene family in soybean [1] and rice [13].
To further analyze whether these tandem or segmental duplication genes are subjected to selection pressure during the evolutionary process, we calculated the Ka and Ks values of these genes.Our results indicate that 95% (35) of duplication genes have a Ka/Ks < 1, and only 2 pairs of duplicate genes have the characteristic of Ka/Ks > 1(Table S4).Similar results have also been found in studies on the evolution of soybean [1], tobacco [15], and Passiflora edulis [19].These results indicated that the PbPOD gene family mainly evolved through positive selection, and positive selection accelerated the evolution of the POD gene family in pears.
As Biłas et al. [20] described that the regulation of gene expression often necessitates the synergy of multiple cisacting elements.Therefore, the identification of cis-elements in PbPOD provided a good opportunity for further understanding the possible transcriptional regulation of these genes in various physiological processes in the future.In our study, a variety of frequently occurring cisacting elements, including MBS, ARE, and ABRE, were investigated in the promoter regions of PbPOD (Fig. 6).In addition, we also found that almost all PbPOD genes contain at least one promoter cis-acting element associated with stress and plant hormones.These results indicated that the PbPOD gene family might be under the regulatory influence of specific plant hormones, potentially playing a role in hormone-driven growth, development, or stress response mechanisms.Similar to our findings, Xiao et al. [21] previously found similar types of cis-acting elements in the POD gene promoter region of grape species in the Rosaceae family.Besides, many previous studies have shown that POD regulates multiple target genes [22], and the loss of their function affects many physiological processes and responses to different plant stresses, leading to phenotypic changes [23,24].In addition, recent studies have also elucidated the role of plant peroxidases in various intracellular mechanisms during plant development and maturation, as well as their response to abiotic and biological environmental pressures [25,26].
RNA-seq data is generally used to study the mRNA expression levels transcribed by specific plant tissues or cells over a certain period of time, and then analyze relevant genes and phenotypes [27].In our research, we used the obtained RNA-seq data from different pear tissues to investigate the possible functions of PbPODs (Fig. 7).Our results found that PbPODs exhibit tissue-specific expression, indicating that PbPODs have different functions.The RNA-seq results showed that among these 113 PbPODs, some genes were not expressed or had low expression levels in the pear tissue.We speculate that these POD genes may play a small role in pear growth and development.In addition, PbPOD26, PbPOD38, PbPOD84, and PbPOD112 were highly expressed in stems of tissues with higher lignin content.The results indicated that those genes may play important roles in pear xylem synthesis.Besides, PbPOD27, PbPOD44, and PbPOD113 were most expressed in pear bud and petal, indicating that they may be related to pear bud extension and flowering formation.Furthermore, some genes were expressed in various tissues, such as PbPOD65, PbPOD83, PbPOD85, and PbPOD87, indicating that these POD genes may have a significant impact on the growth and development process of pears.In summary, our results indicate that the POD family genes play an important regulatory role in the growth and development of pears.
Drought, low temperature, high salinity and other abiotic stresses are serious natural disasters for plants, which seriously affect their growth and development [28].Previous reports on stress treatment have shown that under drought, low temperature, and other stress conditions, the expression of plant POD genes undergoed significant changes [1,19,30,30].However, there is limited research on the response of POD genes to hormones in plants.Previous researches have found that genes associated with hormone stress responses are typically implicated in orchestrating plant stress responses [31,32].These responses are orchestrated through intricate hormone signaling pathways.For instance, studies on PbPOD genes in Pyrus bretschneideri unveiled a plethora of MeJA and ABA-responsive cis-acting elements (Table S5).Moreover, our research unveiled several common cis-acting elements in the POD promoter region, suggesting potential hormone-induced modulation of these genes.In order to illuminate the differential expression patterns of the pear POD genes, we conducted qRT PCR experiments under different hormone treatments (MeJA, ABA, and SA) (Figs. 9, 10, 11 and 12).Among the PbPOD genes, a predominant number of manifested conserved domains, highlighting their potential hormone responsiveness.Interestingly, the sensitivity of PbPOD genes to hormones varied considerably, these treatments strongly upregulated some PbPOD genes, indicating that members of the POD family may have roles in abiotic stress response mechanisms.These results provide evidence that POD members can participate in responses to abiotic stress, particularly hormone stress.We speculated that these three hormones may directly or indirectly regulate the transcription level of pear POD genes.In the future, further studies are imperative to elucidate the precise regulatory influence of these hormones on PbPOD transcription levels.In addition, we also hope to explore the effect of exogenous hormones on the regulation of pear POD expression levels, in order to determine whether the growth and development of pear fruits can be altered by regulating exogenous hormones.
In brief, there is a certain correlation between hormone response and plant resistance to abiotic stress.For example, gene expression patterns related to ethylene suggest that ethylene may indirectly participate in the induction of dormancy genes, thereby enhancing the cold resistance of P. mume [33].Moreover, researchers have also observed this phenomenon in the expression pattern of the TALE gene, where the expression of TALE is not only regulated by certain hormones, but sometimes also influenced by some abiotic stresses [34,35].Aleem's research found that overexpression lines of GsPOD40 exhibit significantly higher drought tolerance compared to wildtype (WT) plants under stress treatment [1].These findings suggest that different POD genes have different functions in various biological processes, including biotic and abiotic stress responses as well as hormone signaling pathways.

Conclusions
This study focused on the Class III peroxidase (POD) family in Chinese pear (Pyrus bretschenedri), an area with limited prior research.The researchers characterized 113 PbPOD genes and categorized them into distinct subfamilies, revealing the role of segmental duplication events in their expansion.GO, KEGG enrichment along cis-acting elements was also performed in pear.The study also examined functional diversity and expression patterns, highlighting the multiple gene responsiveness to stress and their importance in fruit development.The findings position PbPOD genes as promising subjects for further research and potential tools for enhancing fruit quality through molecular breeding.Overall, the study advances our understanding of PODs gene roles in plant development, hormone signaling, and stress responses in the context of Chinese pear.

Identification of POD gene family and analysis physical properties in P. bretschneideri
With the use of the BioEdit tools, we utilized seventythree (73) sequences of Arabidopsis against the pear genome to identify the POD genes with an E-value of 1e − 5 .Moreover, the sequences of pear and Arabidopsis were retrieved from online sources, such as the Pear Genome (http://peargenome.njau.edu.cn)[36] and TAIR genome databases (http://www.arabidopsis.org)[37], respectively.The SMART database (http://smart.embl-heidelberg.de/)[38] and NCBI-Conserved Domain database (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi)were used for the verification of domain composition [3].Sequences with obvious errors in length as well as sequences without POD domains were eliminated before carrying out the analysis.Several physicochemical analyses viz., isoelectronic points (PIs), molecular weight (MW), and GRAVY, were performed for each gene of POD gene by ExPASY PROTPARAM tools (https://web.expasy.org/protparam/)[39].

Sequence alignment and phylogenetic analysis
All POD full-length amino acid sequences of pear and Arabidopsis thaliana were aligned, as well as downloaded from the Arabidopsis (https://www.arabidopsis.org)[7] and pear genome database (http://peargenome.njau.edu.cn/) [40].The MUSCLE was performed using the MEGA 7.0 version for multiple sequences alignment of PODs for phylogenetic analysis.Using the maximum likelihood method (MLM) and phylogenetic tree was constructed, as well the amino acid substitution model was chosen (Jones, Thorton, and Taylor) [41,42].The bootstrap values of one thousand (1000) were used to ensure the reliability of the phylogenetic tree while other parameters were kept as default [43].Finally, the phylogeny tree was constructed through the online itols website (http://itol.embl.de)[44].

GO and KEGG and subcellular localization of PbPOD gene family
The study employed two different online tools, namely the Panther server and the KEGG genome server, to conduct enrichment analyses for Gene Ontology (GO) and KEGG pathways (https://www.genome.jp/kegg/pathway.html) [45][46][47].Subsequently, the pathways that showed enrichment were further examined using TBtools software [48].we further predicted the subcellular localization with the use of the WOLF PSORT (https://wolfpsort.hgc.jp/)online server [49].

Cis-elements predictions of PbPOD gene family
To initiate the analysis, the promoter sequences of POD genes, each spanning 2000 base pairs, were first imported into the CDS sequence from the pear genome.Subsequently, various cis-regulatory elements were identified within each of these promoter sequences using the Plant-Pan database (http://plantpan.itps.ncku.edu.tw/plant-pan4/index.html) [50].

Gene collinearity analysis and chromosomal mapping of PbPOD gene
In this study, the researchers accessed the pear genomic database to determine the chromosomal positions of POD genes and visualized them by TBtool.They then employed this available information to create chromosomal maps for these genes.Furthermore, to analyze the gene collinearity relationship between Pyrus bretschenedri, Prunus avium, Prunus mume, Prunus, and persica, used the Collinearity Scan Toolkit (https://github.com/wyp1125/MCScanX) [51].

Duplication events and calculation of non-synonymous (Ka) and synonymous (Ks)
With the use of the MEGA software (7.0 version), the rate of Ka/Ks was carried out for numbers of the duplicate pairs viz., tandem, dispersed, segmental, and proximal.The method used to find out the ratio of Ks and Ka, followed the Nei-Gojopori method with the bootstrap values of one thousand in MEGA 7.0.The MCScan algorithm (https://github.com/wyp1125/MCScanX)was used to detect the duplication of various types (transposed duplication, dispersed duplication, segmental duplication, and tandem duplication) of POD gene pairs.

Plant material and method
Chinese white pear fruit samples were carefully harvested 39 days after flower (DAF) from Anhui Agricultural University experimental base.To apply specific treatments, melatonin (MEL), salicylic acid (SA), abscisic acid (ABA), and methyl jasmonate (MeJA), a well-documented method described [52] was employed.The treatments salicylic acid (SA), abscisic acid (ABA), and methyl jasmonate (MeJA) were administered at 3 different time points, namely 0 h (control), 1 h, 2 h, and 3 h.As well as melatonin at 0 h (control), 1 h, 4 h, and 16 h.Subsequently, each of the fruit samples was promptly frozen using liquid nitrogen and stored at a temperature of -80 °C to facilitate subsequent in vitro testing.

Transcriptomic data analysis
To do expression profiling, we obtained RNA sequencing data from the NCBI GEO (https://www.ncbi.nlm.nih.gov/geo/) website on stem, sepal, petal, ovary, bud, and leaves were retrieved through the accession numbers SRR8119906, SRR8119889, SRR8119903, SRR8119895, SRR8119898, and SRR8119907.The quantification of expression levels was performed using FPKM (fragments per kilobase of transcript per million fragments mapped).Finally, heat maps were visualized by using the R package.

Isolation of RNA and profiling of the POD gene family in P. bretschneideri
Primer sets meticulously tailored to target specific genes were meticulously designed, and their precision was rigorously assessed using the NCBI Primer Blast tool and Primer Premier 5.0 [53].The comprehensive list of all these primers is shown in Table S1.For consistency and reliability in this study, the pear tubulin gene (AB239680.1)was judiciously chosen to serve as the reference standard [54].Subsequently, cDNA synthesis was accomplished with precision, employing approximately 2 mg of total RNA, utilizing the TransScript® One-Step gDNA Removal and cDNA Synthesis SuperMix, sourced from TRANSGEN in Beijing, China.The quantification of gene expression was executed through qRT-PCR, utilizing the LightCycler 480 SYBRGREEN I Master from Roche, USA, in strict accordance with the protocols outlined in [55], and the manufacturer's instructions.Each individual sample underwent a total of three distinct biological replicates to ensure robust and reliable results.To enable a meaningful comparison with untreated control plants, the gene's relative expression level was meticulously calculated using the 2 −∆∆CT method [56].

Fig. 1
Fig. 1 Phylogenetictree o tree of PODs from Pyrus bretschenedri and Arabidopsis thaliana.The analysis utilized full-length protein sequences of the POD genes and was conducted using the Molecular Evolutionary Genetics Analysis version 7.0 (MEGA 7.0) software.The phylogenetic reconstruction was performed employing the Maximum Likelihood (ML) statistical method.Branch lines of subtrees are colored, indicating different POD subgroups

Fig. 4
Fig.4 Analysis of gene duplication events, microsyntny of the POD gene family in the pear genome.The investigation focused on identifying and characterizing gene duplication events within the POD gene family in the pear (Pyrus bretschenedri) genome.Duplicated gene pairs within the POD gene family are marked by light blue lines, thereby highlighting their genomic locations relative to one another.These light blue lines serve as visual markers that specify duplicated gene pairs, offering a graphical representation of gene duplication events

Fig. 5
Fig. 5 Gene ontology annotation of PbPOD proteins: categorization based on biological process, cellular component and molecular function.The study conducted a comprehensive gene ontology (GO) annotation analysis of PbPOD proteins, aiming to characterize these proteins within the framework of three main GO categories: biological process (BP), cellular component (CC) molecular function (MF).The abscissa of the graphical representation quantifies the proportion of predicted PbPOD proteins that fall under each respective GO term, expressed as a percentage

Fig. 8
Fig. 8 The relative expression of the PbPOD gene response during different development stages of fruit (15 DAF, 39 DAF, 55 DAF, 79 DAF, 102 DAF, and 145 DAF) was measured using qRT-PCR.The results were normalized using an internal control, specifically the tubulin gene.The standard error (SE) represented by the error bars is based on three biological replicates.**Signifcant diference (P < 0.01), *signifcant diference at P < 0.05

Fig. 10 Fig. 9
Fig. 10 The relative expression of the PbPOD gene in response to methyl jasmonate (MeJA) hormonal stress was measured using quantitative PCR (qPCR).The results were normalized using an internal control, specifically the tubulin gene.The standard error (SE) represented by the error bars is based on three biological replicates.**Signifcant diference (P < 0.01), *signifcant diference at P < 0.05